Genomic DNA is constantly challenged by DNA damage either spontaneously induced during cellular metabolism or generated by exogenous DNA damaging agents. During DNA replication, DNA lesions can cause the stalling or collapse of replication forks. Following replication fork damage, activation of the DNA damage signal transduction network promotes the restart of replication forks thereby preserving genomic integrity. We recently identified ZRANB3 as a novel factor that maintains genomic integrity after replication fork damage. In particular, we showed that ZRANB3, an atypical DNA damage enzyme that exhibits both DNA translocase and endonuclease activities, facilitates the restart of replication forks arrested by DNA lesions and protects cells from agents that cause replication fork damage. Despite these important observations, a complete understanding of the precise mechanisms of action exhibited by ZRANB3 to maintain genomic integrity at replication forks is currently lacking. Failure to maintain genomic integrity after replication fork damage results in the accumulation of mutations and genomic rearrangements and predisposes to cancer development. In agreement with a role of ZRANB3 as a putative tumor suppressor, recent cancer genomic studies have reported that ZRANB3 is significantly mutated in endometrial cancer. In addition, mutations predicted to affect the function of ZRANB3 are also found in melanoma and other tumor types. The goal of this proposal is to define the precise biochemical and cellular activities displayed by ZRANB3 for suppressing genomic instability and examine how these activities are affected by ZRANB3 mutations identified in tumors. In particular, we propose: 1) to define the activities exhibited by ZRANB3 for preserving genomic integrity at replication forks; 2) to elucidate the interplay between ZRANB3 and other DNA damage factors that maintain genomic integrity after replication fork damage; 3) to evaluate the contribution of ZRANB3 cancer-associated mutations to genomic instability. Our approach will utilize new in vitro enzymatic assays, innovative proteomic methods, replication fork restart assays on genomic DNA fibers, chromosomal aberration tests and next-generation genome sequencing analyses. We anticipate that our studies will define the unique mechanisms by which ZRANB3 suppresses genomic instability and will provide insights into the potential contribution of ZRANB3 cancer-associated mutations to tumor development.